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1.
Gut Microbes ; 16(1): 2356642, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38769708

RESUMO

Adherent-invasive Escherichia coli (AIEC) strain LF82, isolated from patients with Crohn's disease, invades gut epithelial cells, and replicates in macrophages contributing to chronic inflammation. In this study, we found that RstAB contributing to the colonization of LF82 in a mouse model of chronic colitis by promoting bacterial replication in macrophages. By comparing the transcriptomes of rstAB mutant- and wild-type when infected macrophages, 83 significant differentially expressed genes in LF82 were identified. And we identified two possible RstA target genes (csgD and asr) among the differentially expressed genes. The electrophoretic mobility shift assay and quantitative real-time PCR confirmed that RstA binds to the promoters of csgD and asr and activates their expression. csgD deletion attenuated LF82 intracellular biofilm formation, and asr deletion reduced acid tolerance compared with the wild-type. Acidic pH was shown by quantitative real-time PCR to be the signal sensed by RstAB to activate the expression of csgD and asr. We uncovered a signal transduction pathway whereby LF82, in response to the acidic environment within macrophages, activates transcription of the csgD to promote biofilm formation, and activates transcription of the asr to promote acid tolerance, promoting its replication within macrophages and colonization of the intestine. This finding deepens our understanding of the LF82 replication regulation mechanism in macrophages and offers new perspectives for further studies on AIEC virulence mechanisms.


Assuntos
Aderência Bacteriana , Biofilmes , Infecções por Escherichia coli , Proteínas de Escherichia coli , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Macrófagos , Macrófagos/microbiologia , Animais , Camundongos , Escherichia coli/genética , Escherichia coli/patogenicidade , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Biofilmes/crescimento & desenvolvimento , Infecções por Escherichia coli/microbiologia , Humanos , Concentração de Íons de Hidrogênio , Virulência , Colite/microbiologia , Doença de Crohn/microbiologia , Modelos Animais de Doenças , Transdução de Sinais , Ácidos/metabolismo
3.
Proc Natl Acad Sci U S A ; 121(21): e2400679121, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38753514

RESUMO

Experimental observations tracing back to the 1960s imply that ribosome quantities play a prominent role in determining a cell's growth. Nevertheless, in biologically relevant scenarios, growth can also be influenced by the levels of mRNA and RNA polymerase. Here, we construct a quantitative model of biosynthesis providing testable scenarios for these situations. The model explores a theoretically motivated regime where RNA polymerases compete for genes and ribosomes for transcripts and gives general expressions relating growth rate, mRNA concentrations, ribosome, and RNA polymerase levels. On general grounds, the model predicts how the fraction of ribosomes in the proteome depends on total mRNA concentration and inspects an underexplored regime in which the trade-off between transcript levels and ribosome abundances sets the cellular growth rate. In particular, we show that the model predicts and clarifies three important experimental observations, in budding yeast and Escherichia coli bacteria: i) that the growth-rate cost of unneeded protein expression can be affected by mRNA levels, ii) that resource optimization leads to decreasing trends in mRNA levels at slow growth, and iii) that ribosome allocation may increase, stay constant, or decrease, in response to transcription-inhibiting antibiotics. Since the data indicate that a regime of joint limitation may apply in physiological conditions and not only to perturbations, we speculate that this regime is likely self-imposed.


Assuntos
Escherichia coli , RNA Mensageiro , Ribossomos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Biossíntese de Proteínas , Modelos Biológicos
4.
Proc Natl Acad Sci U S A ; 121(21): e2401748121, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38739789

RESUMO

Potyviridae, the largest family of plant RNA viruses, includes many important pathogens that significantly reduce the yields of many crops worldwide. In this study, we report that the 6-kilodalton peptide 1 (6K1), one of the least characterized potyviral proteins, is an endoplasmic reticulum-localized protein. AI-assisted structure modeling and biochemical assays suggest that 6K1 forms pentamers with a central hydrophobic tunnel, can increase the cell membrane permeability of Escherichia coli and Nicotiana benthamiana, and can conduct potassium in Saccharomyces cerevisiae. An infectivity assay showed that viral proliferation is inhibited by mutations that affect 6K1 multimerization. Moreover, the 6K1 or its homologous 7K proteins from other viruses of the Potyviridae family also have the ability to increase cell membrane permeability and transmembrane potassium conductance. Taken together, these data reveal that 6K1 and its homologous 7K proteins function as viroporins in viral infected cells.


Assuntos
Nicotiana , Nicotiana/virologia , Nicotiana/metabolismo , Potyviridae/genética , Potyviridae/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais/genética , Permeabilidade da Membrana Celular , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Viroporinas/metabolismo , Proteínas Viroporinas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Vírus de Plantas/genética , Vírus de Plantas/fisiologia , Doenças das Plantas/virologia , Potássio/metabolismo
5.
J Pak Med Assoc ; 74(4): 661-665, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38751258

RESUMO

Objectives: To identify various species of non-lactose fermenting gram-negative bacilli involved in urinary tract infections, and to determine their antimicrobial resistance pattern. METHODS: The retrospective, descriptive, cross-sectional study was conducted from January 1 to April 1, 2022, at the Dow University of Health Sciences, Karachi, and comprised data from the institutional diagnostic laboratory that was related to urine samples regardless of age and gender from January 1, 2020, to December 31, 2021. Data was analysed using SPSS version 25. RESULTS: Of the 103,887 urine samples, 41,280(39.7%) were positive, 51,146(49.2%) showed no bacterial growth, 11,000(10.6%) had non-significant bacterial growth and 461(0.4%) had mixed bacterial growth. Of the positive samples, 18359(44.5%) were positive in 2020, and 22,921(55.5%) in 2021. Gram-negative lactose fermenting bacteria included escherichia coli 23,123(22.3%) and klebsiella pneumoniae 2,993(2.9%), gram-negative non-lactose fermenting bacteria included pseudomonas aeruginosa 1,110(1.07%), and gram-positive bacteria included enterococcus 8,008(7.7%). Pseudomonas aeruginosa was most resistant against tobramycin 880(79.3%) and least resistant against piperacillin-tazobactam 146(13%). CONCLUSIONS: Piperacillin-tazobactam was highly sensitive drug against non-lactose fermenting uro-pathogens.


Assuntos
Antibacterianos , Bactérias Gram-Negativas , Infecções Urinárias , Humanos , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções Urinárias/microbiologia , Infecções Urinárias/tratamento farmacológico , Estudos Transversais , Estudos Retrospectivos , Masculino , Feminino , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Adulto , Paquistão , Enterococcus/efeitos dos fármacos , Pessoa de Meia-Idade
6.
Arch Microbiol ; 206(6): 261, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38753095

RESUMO

The search for affordable enzymes with exceptional characteristics is fundamental to overcoming industrial and environmental constraints. In this study, a recombinant GH10 xylanase (Xyn10-HB) from the extremely alkaliphilic bacterium Halalkalibacterium halodurans C-125 cultivated at pH 10 was cloned and expressed in E. coli BL21(DE3). Removal of the signal peptide improved the expression, and an overall activity of 8 U/mL was obtained in the cell-free supernatant. The molecular weight of purified Xyn10-HB was estimated to be 42.6 kDa by SDS-PAGE. The enzyme was active across a wide pH range (5-10) with optimal activity recorded at pH 8.5 and 60 °C. It also presented good stability with a half-life of 3 h under these conditions. Substrate specificity studies showed that Xyn10-HB is a cellulase-free enzyme that conventionally hydrolyse birchwood and oat spelts xylans (Apparent Km of 0.46 mg/mL and 0.54 mg/mL, respectively). HPLC analysis showed that both xylans hydrolysis produced xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from 2 to 9. The conversion yield was 77% after 24 h with xylobiose and xylotriose as the main end-reaction products. When assayed on alkali-extracted wheat straw heteroxylan, the Xyn10-HB produced active XOS with antioxidant activity determined by the DPPH radical scavenging method (IC50 of 0.54 mg/mL after 4 h). Owing to its various characteristics, Xyn10-HB xylanase is a promising candidate for multiple biotechnological applications.


Assuntos
Endo-1,4-beta-Xilanases , Proteínas Recombinantes , Xilanos , Especificidade por Substrato , Hidrólise , Xilanos/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Clonagem Molecular , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Glucuronatos/metabolismo , Estabilidade Enzimática , Cinética , Peso Molecular , Oligossacarídeos/metabolismo , Dissacarídeos
7.
Methods Mol Biol ; 2804: 179-194, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38753148

RESUMO

Antibiotic susceptibility testing (AST) is a routine procedure in diagnostic laboratories to determine pathogen resistance profiles toward antibiotics. The need for fast and accurate resistance results is rapidly increasing with a global rise in pathogen antibiotic resistance over the past years. Microfluidic technologies can enable AST with lower volumes, lower cell numbers, and a reduction in the sample-to-result time compared to state-of-the-art systems. We present a protocol to perform AST on a miniaturized nanoliter chamber array platform. The chambers are filled with antibiotic compounds and oxygen-sensing nanoprobes that serve as a viability indicator. The growth of bacterial cells in the presence of different concentrations of antibiotics is monitored; living cells consume oxygen, which can be observed as an increase of a luminesce signal within the growth chambers. Here, we demonstrate the technique using a quality control Escherichia coli strain, ATCC 35218. The AST requires 20 µL of a diluted bacterial suspension (OD600 = 0.02) and provides resistance profiles about 2-3 h after the inoculation. The microfluidic method can be adapted to other aerobic pathogens and is of particular interest for slow-growing strains.


Assuntos
Antibacterianos , Escherichia coli , Testes de Sensibilidade Microbiana , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/instrumentação , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Oxigênio/metabolismo , Dispositivos Lab-On-A-Chip
8.
J Agric Food Chem ; 72(19): 11029-11040, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38699920

RESUMO

l-Phenylalanine (l-Phe) is widely used in the food and pharmaceutical industries. However, the biosynthesis of l-Phe using Escherichia coli remains challenging due to its lower tolerance to high concentration of l-Phe. In this study, to efficiently synthesize l-Phe, the l-Phe biosynthetic pathway was reconstructed by expressing the heterologous genes aroK1, aroL1, and pheA1, along with the native genes aroA, aroC, and tyrB in the shikimate-producing strain E. coli SA09, resulting in the engineered strain E. coli PHE03. Subsequently, adaptive evolution was conducted on E. coli PHE03 to enhance its tolerance to high concentrations of l-Phe, resulting in the strain E. coli PHE04, which reduced the cell mortality to 36.2% after 48 h of fermentation. To elucidate the potential mechanisms, transcriptional profiling was conducted, revealing MarA, a DNA-binding transcriptional dual regulator, as playing a crucial role in enhancing cell membrane integrity and fluidity for improving cell tolerance to high concentrations of l-Phe. Finally, the titer, yield, and productivity of l-Phe with E. coli PHE05 overexpressing marA were increased to 80.48 g/L, 0.27 g/g glucose, and 1.68 g/L/h in a 5-L fed-batch fermentation, respectively.


Assuntos
Escherichia coli , Fermentação , Engenharia Metabólica , Fenilalanina , Escherichia coli/genética , Escherichia coli/metabolismo , Fenilalanina/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Vias Biossintéticas
9.
Biochem Biophys Res Commun ; 716: 150009, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38697010

RESUMO

The SOS response is a condition that occurs in bacterial cells after DNA damage. In this state, the bacterium is able to reсover the integrity of its genome. Due to the increased level of mutagenesis in cells during the repair of DNA double-strand breaks, the SOS response is also an important mechanism for bacterial adaptation to the antibiotics. One of the key proteins of the SOS response is the SMC-like protein RecN, which helps the RecA recombinase to find a homologous DNA template for repair. In this work, the localization of the recombinant RecN protein in living Escherichia coli cells was revealed using fluorescence microscopy. It has been shown that the RecN, outside the SOS response, is predominantly localized at the poles of the cell, and in dividing cells, also localized at the center. Using in vitro methods including fluorescence microscopy and optical tweezers, we show that RecN predominantly binds single-stranded DNA in an ATP-dependent manner. RecN has both intrinsic and single-stranded DNA-stimulated ATPase activity. The results of this work may be useful for better understanding of the SOS response mechanism and homologous recombination process.


Assuntos
DNA Bacteriano , Escherichia coli , Microscopia de Fluorescência , Imagem Individual de Molécula , Microscopia de Fluorescência/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Imagem Individual de Molécula/métodos , DNA Bacteriano/metabolismo , DNA Bacteriano/genética , Resposta SOS em Genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Ligação Proteica , Recombinases Rec A/metabolismo , Recombinases Rec A/genética , Pinças Ópticas
10.
Proc Natl Acad Sci U S A ; 121(21): e2401738121, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38743623

RESUMO

Studies have determined that nonredox enzymes that are cofactored with Fe(II) are the most oxidant-sensitive targets inside Escherichia coli. These enzymes use Fe(II) cofactors to bind and activate substrates. Because of their solvent exposure, the metal can be accessed and oxidized by reactive oxygen species, thereby inactivating the enzyme. Because these enzymes participate in key physiological processes, the consequences of stress can be severe. Accordingly, when E. coli senses elevated levels of H2O2, it induces both a miniferritin and a manganese importer, enabling the replacement of the iron atom in these enzymes with manganese. Manganese does not react with H2O2 and thereby preserves enzyme activity. In this study, we examined several diverse microbes to identify the metal that they customarily integrate into ribulose-5-phosphate 3-epimerase, a representative of this enzyme family. The anaerobe Bacteroides thetaiotaomicron, like E. coli, uses iron. In contrast, Bacillus subtilis and Lactococcus lactis use manganese, and Saccharomyces cerevisiae uses zinc. The latter organisms are therefore well suited to the oxidizing environments in which they dwell. Similar results were obtained with peptide deformylase, another essential enzyme of the mononuclear class. Strikingly, heterologous expression experiments show that it is the metal pool within the organism, rather than features of the protein itself, that determine which metal is incorporated. Further, regardless of the source organism, each enzyme exhibits highest turnover with iron and lowest turnover with zinc. We infer that the intrinsic catalytic properties of the metal cannot easily be retuned by evolution of the polypeptide.


Assuntos
Escherichia coli , Ferro , Manganês , Manganês/metabolismo , Ferro/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Peróxido de Hidrogênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Zinco/metabolismo , Lactococcus lactis/enzimologia , Lactococcus lactis/metabolismo , Oxirredução , Metais/metabolismo
11.
Biochim Biophys Acta Gene Regul Mech ; 1867(2): 195032, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38692564

RESUMO

Small non-coding 6S RNA mimics DNA promoters and binds to the σ70 holoenzyme of bacterial RNA polymerase (RNAP) to suppress transcription of various genes mainly during the stationary phase of cell growth or starvation. This inhibition can be relieved upon synthesis of short product RNA (pRNA) performed by RNAP from the 6S RNA template. Here, we have shown that pRNA synthesis depends on specific contacts of 6S RNA with RNAP and interactions of the σ finger with the RNA template in the active site of RNAP, and is also modulated by the secondary channel factors. We have adapted a molecular beacon assay with fluorescently labeled σ70 to analyze 6S RNA release during pRNA synthesis. We found the kinetics of 6S RNA release to be oppositely affected by mutations in the σ finger and in the CRE pocket of core RNAP, similarly to the reported role of these regions in promoter-dependent transcription. Secondary channel factors, DksA and GreB, inhibit pRNA synthesis and 6S RNA release from RNAP, suggesting that they may contribute to the 6S RNA-mediated switch in transcription during stringent response. Our results demonstrate that pRNA synthesis depends on a similar set of contacts between RNAP and 6S RNA as in the case of promoter-dependent transcription initiation and reveal that both processes can be regulated by universal transcription factors acting on RNAP.


Assuntos
RNA Polimerases Dirigidas por DNA , Proteínas de Escherichia coli , RNA Bacteriano , Fator sigma , Transcrição Gênica , RNA Polimerases Dirigidas por DNA/metabolismo , Fator sigma/metabolismo , Fator sigma/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , RNA Bacteriano/metabolismo , RNA Bacteriano/genética , Regiões Promotoras Genéticas , RNA não Traduzido/metabolismo , RNA não Traduzido/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Ligação Proteica , Fatores de Elongação da Transcrição
12.
ACS Synth Biol ; 13(5): 1434-1441, 2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38695987

RESUMO

Enzymatic cascades have become a green and sustainable approach for the synthesis of valuable chemicals and pharmaceuticals. Using sequential enzymes to construct a multienzyme complex is an effective way to enhance the overall performance of biosynthetic routes. Here we report the design of an efficient in vitro hybrid biocatalytic system by assembling three enzymes that can convert styrene to (S)-1-phenyl-1,2-ethanediol. Specifically, we prepared the three enzymes in different ways, which were cell surface-displayed, purified, and cell-free expressed. To assemble them, we fused two orthogonal peptide-protein pairs (i.e., SpyTag/SpyCatcher and SnoopTag/SnoopCatcher) to the three enzymes, allowing their spatial organization by covalent assembly. By doing this, we constructed a multienzyme complex, which could enhance the production of (S)-1-phenyl-1,2-ethanediol by 3 times compared to the free-floating enzyme system without assembly. After optimization of the reaction system, the final product yield reached 234.6 µM with a substrate conversion rate of 46.9% (based on 0.5 mM styrene). Taken together, our strategy integrates the merits of advanced biochemical engineering techniques, including cellular surface display, spatial enzyme organization, and cell-free expression, which offers a new solution for chemical biosynthesis by enzymatic cascade biotransformation. We, therefore, anticipate that our approach will hold great potential for designing and constructing highly efficient systems to synthesize chemicals of agricultural, industrial, and pharmaceutical significance.


Assuntos
Biocatálise , Sistema Livre de Células , Estireno/metabolismo , Estireno/química , Escherichia coli/genética , Escherichia coli/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo
13.
ACS Synth Biol ; 13(5): 1572-1581, 2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38717981

RESUMO

Inside cells, various biological systems work cooperatively for homeostasis and self-replication. These systems do not work independently as they compete for shared elements like ATP and NADH. However, it has been believed that such competition is not a problem in codependent biological systems such as the energy-supplying glycolysis and the energy-consuming translation system. In this study, we biochemically reconstituted the coupling system of glycolysis and translation using purified elements and found that the competition for ATP between glycolysis and protein synthesis interferes with their coupling. Both experiments and simulations revealed that this interference is derived from a metabolic tug-of-war between glycolysis and translation based on their reaction rates, which changes the threshold of the initial substrate concentration for the success coupling. By the metabolic tug-of-war, translation energized by strong glycolysis is facilitated by an exogenous ATPase, which normally inhibits translation. These findings provide chemical insights into the mechanism of competition among biological systems in living cells and provide a framework for the construction of synthetic metabolism in vitro.


Assuntos
Trifosfato de Adenosina , Glicólise , Biossíntese de Proteínas , Trifosfato de Adenosina/metabolismo , NAD/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética
14.
World J Microbiol Biotechnol ; 40(6): 183, 2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38722449

RESUMO

Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6 H-S-PaLPL) works optimally at 35 °C and pH 6.5 with remarkable thermostability at 35-95 °C. Thermo-inactivation kinetics of 6 H-S-PaLPL were also studied at 35-95 °C with first order rate constant (kIN) of 5.58 × 10- 2 h-1 and half-life of 12 ± 0 h at 95 °C. Km and Vmax for the hydrolysis of 4-nitrophenyl butyrate were calculated to be 2 ± 0.015 mM and 3882 ± 22.368 U/mg, respectively. 2.4-fold increase in Vmax of Pa-LPL was observed after fusion of 6xHis-SUMO tag to its N-terminal. It is the first report on the utilization of SUMO fusion tag to enhance the overall stability and activity of Pa-LPL. Fusion of 6xHis-SUMO tag not only aided in the purification process but also played a crucial role in increasing the thermostability and activity of the enzyme. SUMO-fused enzyme, thus generated, can serve as an important candidate for degumming of vegetable oils at industrial scale.


Assuntos
Estabilidade Enzimática , Escherichia coli , Pyrococcus abyssi , Proteínas Recombinantes de Fusão , Temperatura , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Pyrococcus abyssi/genética , Pyrococcus abyssi/enzimologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Vetores Genéticos/metabolismo , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Proteína SUMO-1/química , Clonagem Molecular , Solubilidade
15.
Microb Cell Fact ; 23(1): 130, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38711033

RESUMO

BACKGROUND: Cyclic ß-1,2-glucans (CßG) are bacterial cyclic homopolysaccharides with interesting biotechnological applications. These ring-shaped molecules have a hydrophilic surface that confers high solubility and a hydrophobic cavity able to include poorly soluble molecules. Several studies demonstrate that CßG and many derivatives can be applied in drug solubilization and stabilization, enantiomer separation, catalysis, synthesis of nanomaterials and even as immunomodulators, suggesting these molecules have great potential for their industrial and commercial exploitation. Nowadays, there is no method to produce CßG by chemical synthesis and bacteria that synthesize them are slow-growing or even pathogenic, which makes the scaling up of the process difficult and expensive. Therefore, scalable production and purification methods are needed to afford the demand and expand the repertoire of applications of CßG. RESULTS: We present the production of CßG in specially designed E. coli strains by means of the deletion of intrinsic polysaccharide biosynthetic genes and the heterologous expression of enzymes involved in CßG synthesis, transport and succinilation. These strains produce different types of CßG: unsubstituted CßG, anionic CßG and CßG of high size. Unsubstituted CßG with a degree of polymerization of 17 to 24 glucoses were produced and secreted to the culture medium by one of the strains. Through high cell density culture (HCDC) of that strain we were able to produce 4,5 g of pure unsubstituted CßG /L in culture medium within 48 h culture. CONCLUSIONS: We have developed a new recombinant bacterial system for the synthesis of cyclic ß-1,2-glucans, expanding the use of bacteria as a platform for the production of new polysaccharides with biotechnological applications. This new approach allowed us to produce CßG in E. coli with high yields and the highest volumetric productivity reported to date. We expect this new highly scalable system facilitates CßG availability for further research and the widespread use of these promising molecules across many application fields.


Assuntos
Escherichia coli , beta-Glucanas , Escherichia coli/metabolismo , Escherichia coli/genética , beta-Glucanas/metabolismo
16.
Microb Cell Fact ; 23(1): 132, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38711050

RESUMO

BACKGROUND: 1,5-pentanediol (1,5-PDO) is a linear diol with an odd number of methylene groups, which is an important raw material for polyurethane production. In recent years, the chemical methods have been predominantly employed for synthesizing 1,5-PDO. However, with the increasing emphasis on environmentally friendly production, it has been a growing interest in the biosynthesis of 1,5-PDO. Due to the limited availability of only three reported feasible biosynthesis pathways, we developed a new biosynthetic pathway to form a cell factory in Escherichia coli to produce 1,5-PDO. RESULTS: In this study, we reported an artificial pathway for the synthesis of 1,5-PDO from lysine with an integrated cofactor and co-substrate recycling and also evaluated its feasibility in E.coli. To get through the pathway, we first screened aminotransferases originated from different organisms to identify the enzyme that could successfully transfer two amines from cadaverine, and thus GabT from E. coli was characterized. It was then cascaded with lysine decarboxylase and alcohol dehydrogenase from E. coli to achieve the whole-cell production of 1,5-PDO from lysine. To improve the whole-cell activity for 1,5-PDO production, we employed a protein scaffold of EutM for GabT assembly and glutamate dehydrogenase was also validated for the recycling of NADPH and α-ketoglutaric acid (α-KG). After optimizing the cultivation and bioconversion conditions, the titer of 1,5-PDO reached 4.03 mM. CONCLUSION: We established a novel pathway for 1,5-PDO production through two consecutive transamination reaction from cadaverine, and also integrated cofactor and co-substrate recycling system, which provided an alternative option for the biosynthesis of 1,5-PDO.


Assuntos
Vias Biossintéticas , Escherichia coli , Escherichia coli/metabolismo , Escherichia coli/genética , Engenharia Metabólica/métodos , Glicóis/metabolismo , Lisina/metabolismo , Lisina/biossíntese , Álcool Desidrogenase/metabolismo , Transaminases/metabolismo , Transaminases/genética , Carboxiliases/metabolismo
17.
Biotechnol J ; 19(5): e2300581, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38719587

RESUMO

Human interleukin-3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. However, its production in Escherichia coli has historically been challenging due to its aggregation into inclusion bodies, requiring intricate solubilization and refolding procedures. This study introduces an innovative approach employing two chaperone proteins, maltose binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a'), as N-terminal fusion tags. Histidine tag (H) was added at the beginning of each chaperone protein gene for easy purification. This fusion of chaperone proteins significantly improved IL3 solubility across various E. coli strains and temperature conditions, eliminating the need for laborious refolding procedures. Following expression optimization, H-PDIb'a'-IL3 was purified using two chromatographic methods, and the subsequent removal of the H-PDIb'a' tag yielded high-purity IL3. The identity of the purified protein was confirmed through liquid chromatography coupled with tandem mass spectrometry analysis. Biological activity assays using human erythroleukemia TF-1 cells revealed a unique two-step stimulation pattern for both purified IL3 and the H-PDIb'a'-IL3 fusion protein, underscoring the protein's functional integrity and revealing novel insights into its cellular interactions. This study advances the understanding of IL3 expression and activity while introducing novel considerations for protein fusion strategies.


Assuntos
Escherichia coli , Interleucina-3 , Isomerases de Dissulfetos de Proteínas , Proteínas Recombinantes de Fusão , Humanos , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Interleucina-3/metabolismo , Interleucina-3/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Linhagem Celular Tumoral , Solubilidade
18.
Biotechnol J ; 19(5): e2400023, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38719589

RESUMO

The discovery of antibiotics has noticeably promoted the development of human civilization; however, antibiotic resistance in bacteria caused by abusing and overusing greatly challenges human health and food safety. Considering the worsening situation, it is an urgent demand to develop emerging nontraditional technologies or methods to address this issue. With the expanding of synthetic biology, optogenetics exhibits a tempting prospect for precisely regulating gene expression in many fields. Consequently, it is attractive to employ optogenetics to reduce the risk of antibiotic resistance. Here, a blue light-controllable gene expression system was established in Escherichia coli based on a photosensitive DNA-binding protein (EL222). Further, this strategy was successfully applied to repress the expression of ß-lactamase gene (bla) using blue light illumination, resulting a dramatic reduction of ampicillin resistance in engineered E. coli. Moreover, blue light was utilized to induce the expression of the mechanosensitive channel of large conductance (MscL), triumphantly leading to the increase of streptomycin susceptibility in engineered E. coli. Finally, the increased susceptibility of ampicillin and streptomycin was simultaneously induced by blue light in the same E. coli cell, revealing the excellent potential of this strategy in controlling multidrug-resistant (MDR) bacteria. As a proof of concept, our work demonstrates that light can be used as an alternative tool to prolong the use period of common antibiotics without developing new antibiotics. And this novel strategy based on optogenetics shows a promising foreground to combat antibiotic resistance in the future.


Assuntos
Antibacterianos , Escherichia coli , Luz , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Antibacterianos/farmacologia , Optogenética/métodos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ampicilina/farmacologia , beta-Lactamases/genética , beta-Lactamases/metabolismo , Farmacorresistência Bacteriana/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Estreptomicina/farmacologia , Luz Azul
19.
Biotechnol J ; 19(5): e2300664, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38719620

RESUMO

CYP116B5 is a class VII P450 in which the heme domain is linked to a FMN and 2Fe2S-binding reductase. Our laboratory has proved that the CYP116B5 heme domain (CYP116B5-hd) is capable of catalyzing the oxidation of substrates using H2O2. Recently, the Molecular Lego approach was applied to join the heme domain of CYP116B5 to sarcosine oxidase (SOX), which provides H2O2 in-situ by the sarcosine oxidation. In this work, the chimeric self-sufficient fusion enzyme CYP116B5-SOX was heterologously expressed, purified, and characterized for its functionality by absorbance and fluorescence spectroscopy. Differential scanning calorimetry (DSC) experiments revealed a TM of 48.4 ± 0.04 and 58.3 ± 0.02°C and a enthalpy value of 175,500 ± 1850 and 120,500 ± 1350 cal mol-1 for the CYP116B5 and SOX domains respectively. The fusion enzyme showed an outstanding chemical stability in presence of up to 200 mM sarcosine or 5 mM H2O2 (4.4 ± 0.8 and 11.0 ± 2.6% heme leakage respectively). Thanks to the in-situ H2O2 generation, an improved kcat/KM for the p-nitrophenol conversion was observed (kcat of 20.1 ± 0.6 min-1 and KM of 0.23 ± 0.03 mM), corresponding to 4 times the kcat/KM of the CYP116B5-hd. The aim of this work is the development of an engineered biocatalyst to be exploited in bioremediation. In order to tackle this challenge, an E. coli strain expressing CYP116B5-SOX was employed to exploit this biocatalyst for the oxidation of the wastewater contaminating-drug tamoxifen. Data show a 12-fold increase in tamoxifen N-oxide production-herein detected for the first time as CYP116B5 metabolite-compared to the direct H2O2 supply, equal to the 25% of the total drug conversion.


Assuntos
Biodegradação Ambiental , Sistema Enzimático do Citocromo P-450 , Escherichia coli , Peróxido de Hidrogênio , Sarcosina Oxidase , Peróxido de Hidrogênio/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Sarcosina Oxidase/metabolismo , Sarcosina Oxidase/genética , Sarcosina Oxidase/química , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/química , Oxirredução , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/química , Sarcosina/metabolismo , Sarcosina/análogos & derivados
20.
Protein Sci ; 33(6): e5001, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38723111

RESUMO

De novo protein design expands the protein universe by creating new sequences to accomplish tailor-made enzymes in the future. A promising topology to implement diverse enzyme functions is the ubiquitous TIM-barrel fold. Since the initial de novo design of an idealized four-fold symmetric TIM barrel, the family of de novo TIM barrels is expanding rapidly. Despite this and in contrast to natural TIM barrels, these novel proteins lack cavities and structural elements essential for the incorporation of binding sites or enzymatic functions. In this work, we diversified a de novo TIM barrel by extending multiple ßα-loops using constrained hallucination. Experimentally tested designs were found to be soluble upon expression in Escherichia coli and well-behaved. Biochemical characterization and crystal structures revealed successful extensions with defined α-helical structures. These diversified de novo TIM barrels provide a framework to explore a broad spectrum of functions based on the potential of natural TIM barrels.


Assuntos
Modelos Moleculares , Escherichia coli/genética , Escherichia coli/metabolismo , Cristalografia por Raios X , Dobramento de Proteína , Engenharia de Proteínas/métodos , Proteínas/química , Proteínas/metabolismo
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